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NAR Genomics and Bioinformatics Jun 2023DNA methylation, the addition of a methyl (CH) group to a cytosine residue, is an evolutionarily conserved epigenetic mark involved in a number of different biological...
DNA methylation, the addition of a methyl (CH) group to a cytosine residue, is an evolutionarily conserved epigenetic mark involved in a number of different biological functions in eukaryotes, including transcriptional regulation, chromatin structural organization, cellular differentiation and development. In the social amoeba , previous studies have shown the existence of a DNA methyltransferase (DNMA) belonging to the DNMT2 family, but the extent and function of 5-methylcytosine in the genome are unclear. Here, we present the whole genome DNA methylation profile of using deep coverage replicate sequencing of bisulfite-converted gDNA extracted from post-starvation cells. We find an overall very low number of sites with any detectable level of DNA methylation, occurring at significant levels in only 303-3432 cytosines out of the ∼7.5 million total cytosines in the genome depending on the replicate. Furthermore, a knockout of the DNMA enzyme leads to no overall decrease in DNA methylation. Of the identified sites, significant methylation is only detected at 11 sites in all four of the methylomes analyzed. Targeted bisulfite PCR sequencing and computational analysis demonstrate that the methylation profile does not change during development and that these 11 cytosines are most likely false positives generated by protection from bisulfite conversion due to their location in hairpin-forming palindromic DNA sequences. Our data therefore provide evidence that there is no significant DNA methylation in before fruiting body formation and identify a reproducible experimental artifact from bisulfite sequencing.
PubMed: 37081864
DOI: 10.1093/nargab/lqad035 -
ELife Apr 2023Cells are perpetually challenged by pathogens, protein aggregates or chemicals, that induce plasma membrane or endolysosomal compartments damage. This severe stress is...
Cells are perpetually challenged by pathogens, protein aggregates or chemicals, that induce plasma membrane or endolysosomal compartments damage. This severe stress is recognised and controlled by the endosomal sorting complex required for transport (ESCRT) and the autophagy machineries, which are recruited to damaged membranes to either repair or to remove membrane remnants. Yet, insight is limited about how damage is sensed and which effectors lead to extensive tagging of the damaged organelles with signals, such as K63-polyubiquitin, required for the recruitment of membrane repair or removal machineries. To explore the key factors responsible for detection and marking of damaged compartments, we use the professional phagocyte . We found an evolutionary conserved E3-ligase, TrafE, that is robustly recruited to intracellular compartments disrupted after infection with or after sterile damage caused by chemical compounds. TrafE acts at the intersection of ESCRT and autophagy pathways and plays a key role in functional recruitment of the ESCRT subunits ALIX, Vps32 and Vps4 to damage sites. Importantly, we show that the absence of TrafE severely compromises the xenophagy restriction of mycobacteria as well as ESCRT-mediated and autophagy-mediated endolysosomal membrane damage repair, resulting in early cell death.
Topics: Mycobacterium marinum; Endosomal Sorting Complexes Required for Transport; Dictyostelium; Endosomes; Autophagy
PubMed: 37070811
DOI: 10.7554/eLife.85727 -
Journal of Pharmacological Sciences May 2023Differentiation-inducing factor 1 (DIF-1) is a morphogen produced by Dictyostelium discoideum that inhibits the proliferation and migration of both D. discoideum and...
Differentiation-inducing factor 1 (DIF-1) is a morphogen produced by Dictyostelium discoideum that inhibits the proliferation and migration of both D. discoideum and most mammalian cells. Herein, we assessed the effect of DIF-1 on mitochondria, because DIF-3, which is similar to DIF-1, reportedly localizes in the mitochondria when added exogenously, however the significance of this localization remains unclear. Cofilin is an actin depolymerization factor that is activated by dephosphorylation at Ser-3. By regulating the actin cytoskeleton, cofilin induces mitochondrial fission, the first step in mitophagy. Here, we report that DIF-1 activates cofilin and induces mitochondrial fission and mitophagy mainly using human umbilical vein endothelial cells (HUVECs). AMP-activated kinase (AMPK), a downstream molecule of DIF-1 signaling, is required for cofilin activation. Pyridoxal phosphatase (PDXP)-known to directly dephosphorylate cofilin-is also required for the effect of DIF-1 on cofilin, indicating that DIF-1 activates cofilin through AMPK and PDXP. Cofilin knockdown inhibits mitochondrial fission and decreases mitofusin 2 (Mfn2) protein levels, a hallmark of mitophagy. Taken together, these results indicate that cofilin is required for DIF-1- induced mitochondrial fission and mitophagy.
Topics: Animals; Humans; AMP-Activated Protein Kinases; Actin Depolymerizing Factors; Mitochondrial Dynamics; Dictyostelium; Endothelial Cells; Cell Differentiation; Phosphoric Monoester Hydrolases; Pyridoxal; Hexanones; Mammals
PubMed: 37059490
DOI: 10.1016/j.jphs.2023.02.009 -
The Growing Liberality Observed in Primary Animal and Plant Cultures is Common to the Social Amoeba.Journal of Bioinformatics and Systems... 2023Tissue culture environment liberates cells fromordinary laws of multi-cellular organisms. This liberation enables cells several behaviors, such as proliferation,...
Tissue culture environment liberates cells fromordinary laws of multi-cellular organisms. This liberation enables cells several behaviors, such as proliferation, dedifferentiation, acquisition of pluripotency, immortalization, and reprogramming. Recently, the quantitative value of cellular dedifferentiation and differentiation was defined as "liberality", which is measurable as Shannon entropy of numerical transcriptome data and Lempel-Zip complexity of nucleotide sequence transcriptome data. The increasing liberality induced by the culture environment had first been observed in animal cells and had reconfirmed in plant cells.The phenomena may be common across the kingdom, also in a social amoeba. We measured the liberality of the social amoeba which disaggregated from multicellular aggregates and transferred into a liquid medium.
PubMed: 37033694
DOI: 10.26502/jbsb.5107045 -
The Journal of Cell Biology Jun 2023The actin-rich cortex plays a fundamental role in many cellular processes. Its architecture and molecular composition vary across cell types and physiological states....
The actin-rich cortex plays a fundamental role in many cellular processes. Its architecture and molecular composition vary across cell types and physiological states. The full complement of actin assembly factors driving cortex formation and how their activities are spatiotemporally regulated remain to be fully elucidated. Using Dictyostelium as a model for polarized and rapidly migrating cells, we show that GxcM, a RhoGEF localized specifically in the rear of migrating cells, functions together with F-BAR protein Fbp17, a small GTPase RacC, and the actin nucleation-promoting factor WASP to coordinately promote Arp2/3 complex-mediated cortical actin assembly. Overactivation of this signaling cascade leads to excessive actin polymerization in the rear cortex, whereas its disruption causes defects in cortical integrity and function. Therefore, apart from its well-defined role in the formation of the protrusions at the cell front, the Arp2/3 complex-based actin carries out a previously unappreciated function in building the rear cortical subcompartment in rapidly migrating cells.
Topics: Actin-Related Protein 2-3 Complex; Actins; Dictyostelium; Signal Transduction; Wiskott-Aldrich Syndrome Protein; Protozoan Proteins
PubMed: 37010470
DOI: 10.1083/jcb.202208151 -
Journal of Biological Methods 2023To fully understand any cellular process, we not only need to identify the proteins implicated, but also how the protein network is structurally and spatially organized...
To fully understand any cellular process, we not only need to identify the proteins implicated, but also how the protein network is structurally and spatially organized and changes over time. However, the dynamic nature of many protein interactions involved in cellular signaling pathways continues to be the bottleneck in mapping and studying protein networks. Fortunately, a recently developed proximity labeling method using engineered ascorbic acid peroxidase 2 (APEX2) in mammalian cells allows the identification of weak and/or transient protein interactions with spatial and temporal resolution. Here, we describe a protocol for successfully using the APEX2-proximity labeling method in , using the cAMP receptor cAR1 as example. Coupled to the identification of the labeled proteins by mass spectrometry, this method expands proteomics toolbox and should be widely useful for identifying interacting partners involved in a variety of biological processes in .
PubMed: 37007980
DOI: 10.14440/jbm.2023.396 -
Current Biology : CB May 2023Negative chemotaxis, where eukaryotic cells migrate away from repellents, is important throughout biology, for example, in nervous system patterning and resolution of...
Negative chemotaxis, where eukaryotic cells migrate away from repellents, is important throughout biology, for example, in nervous system patterning and resolution of inflammation. However, the mechanisms by which molecules repel migrating cells are unknown. Here, we use predictive modeling and experiments with Dictyostelium cells to show that competition between different ligands that bind to the same receptor leads to effective chemorepulsion. 8-CPT-cAMP, widely described as a simple chemorepellent, is inactive on its own and only repels cells when it acts in combination with the attractant cAMP. If cells degrade either competing ligand, the pattern of migration becomes more complex; cells may be repelled in one part of a gradient but attracted elsewhere, leading to populations moving in different directions in the same assay or converging in an arbitrary place. More counterintuitively still, two chemicals that normally attract cells can become repellent when combined. Computational models of chemotaxis are now accurate enough to predict phenomena that have not been anticipated by experiments. We have used them to identify new mechanisms that drive reverse chemotaxis, which we have confirmed through experiments with real cells. These findings are important whenever multiple ligands compete for the same receptors.
Topics: Chemotaxis; Chemotactic Factors; Dictyostelium; Eukaryotic Cells
PubMed: 37001521
DOI: 10.1016/j.cub.2023.03.006 -
ACS Nano May 2023Metal-induced energy transfer (MIET) imaging is an easy-to-implement super-resolution modality that achieves nanometer resolution along the optical axis of a microscope....
Metal-induced energy transfer (MIET) imaging is an easy-to-implement super-resolution modality that achieves nanometer resolution along the optical axis of a microscope. Although its capability in numerous biological and biophysical studies has been demonstrated, its implementation for live-cell imaging with fluorescent proteins is still lacking. Here, we present its applicability and capabilities for live-cell imaging with fluorescent proteins in diverse cell types (adult human stem cells, human osteo-sarcoma cells, and cells), and with various fluorescent proteins (GFP, mScarlet, RFP, YPet). We show that MIET imaging achieves nanometer axial mapping of living cellular and subcellular components across multiple time scales, from a few milliseconds to hours, with negligible phototoxic effects.
Topics: Humans; Microscopy, Fluorescence; Dictyostelium; Energy Transfer; Fluorescent Dyes
PubMed: 36995274
DOI: 10.1021/acsnano.2c12372 -
International Journal of Molecular... Mar 2023is a soil-dwelling unicellular eukaryote that accumulates extracellular polyphosphate (polyP). At high cell densities, when the cells are about to overgrow their food...
is a soil-dwelling unicellular eukaryote that accumulates extracellular polyphosphate (polyP). At high cell densities, when the cells are about to overgrow their food supply and starve, the corresponding high extracellular concentrations of polyP allow the cells to preemptively anticipate starvation, inhibit proliferation, and prime themselves to begin development. In this report, we show that starved cells accumulate cell surface and extracellular polyP. Starvation reduces macropinocytosis, exocytosis, and phagocytosis, and we find that these effects require the G protein-coupled polyP receptor (GrlD) and two enzymes, Polyphosphate kinase 1 (Ppk1), which is required for synthesizing intracellular polyP, cell surface polyP, and some of the extracellular polyP, and Inositol hexakisphosphate kinase (I6kA), which is required for cell surface polyP and polyP binding to cells, and some of the extracellular polyP. PolyP reduces membrane fluidity, and we find that starvation reduces membrane fluidity; this effect requires GrlD and Ppk1, but not I6kA. Together, these data suggest that in starved cells, extracellular polyP decreases membrane fluidity, possibly as a protective measure. In the starved cells, sensing polyP appears to decrease energy expenditure from ingestion, and decrease exocytosis, and to both decrease energy expenditures and retain nutrients.
Topics: Dictyostelium; Polyphosphates; Receptors, G-Protein-Coupled; Phagocytosis; Exocytosis
PubMed: 36982997
DOI: 10.3390/ijms24065923 -
European Journal of Cell Biology Jun 2023The neuronal ceroid lipofuscinoses (NCLs), collectively referred to as Batten disease, are a group of fatal neurodegenerative disorders that primarily affect children.... (Review)
Review
The neuronal ceroid lipofuscinoses (NCLs), collectively referred to as Batten disease, are a group of fatal neurodegenerative disorders that primarily affect children. The etiology of Batten disease is linked to mutations in 13 genes that encode distinct CLN proteins, whose functions have yet to be fully elucidated. The social amoeba Dictyostelium discoideum has been adopted as an efficient and powerful model system for studying the diverse cellular roles of CLN proteins. The genome of D. discoideum encodes several homologs of human CLN proteins, and a growing body of literature supports the conserved roles and networking of CLN proteins in D. discoideum and humans. In humans, CLN proteins have diverse cellular roles related to autophagy, signal transduction, lipid homeostasis, lysosomal ion homeostasis, and intracellular trafficking. Recent work also indicates that CLN proteins play an important role in protein secretion. Remarkably, many of these findings have found parallels in studies with D. discoideum. Accordingly, this review will highlight the translatable value of novel work with D. discoideum in the field of NCL research and propose further avenues of research using this biomedical model organism for studying the NCLs.
Topics: Child; Humans; Dictyostelium; Neuronal Ceroid-Lipofuscinoses; Proteins; Lysosomes; Mutation
PubMed: 36917916
DOI: 10.1016/j.ejcb.2023.151305